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Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

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In vitro recycling of nuclear GRs. GrH2 cells grown on  coverslips were treated with corticosterone (Cort) for 1 h, and  then withdrawn from hormone for 30 min. Cells were then permeabilized and intact nuclei were incubated with 50 μl transport  mixture containing HeLa cytosol, ATP, and an ATP-regenerating system for 20 min at 30°C, with (C and D) or without (A and  B) 1 μM corticosterone present. Nuclei were then either directly  fixed without extraction (A and C) or extracted with Hypo buffer  before fixation (B and D). GRs were detected by IIF.
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Figure 5: In vitro recycling of nuclear GRs. GrH2 cells grown on coverslips were treated with corticosterone (Cort) for 1 h, and then withdrawn from hormone for 30 min. Cells were then permeabilized and intact nuclei were incubated with 50 μl transport mixture containing HeLa cytosol, ATP, and an ATP-regenerating system for 20 min at 30°C, with (C and D) or without (A and B) 1 μM corticosterone present. Nuclei were then either directly fixed without extraction (A and C) or extracted with Hypo buffer before fixation (B and D). GRs were detected by IIF.

Mentions: Since GR nuclear export is extremely slow, we did not expect considerable redistribution of receptors from the nucleus to the cytoplasm during the 20-min hormone withdrawal and 10-min secondary hormone treatment. However, to exclude the possibility that the redistribution of nuclear GRs under our limited hormone withdrawal (i.e., 20 min) was proceeding undetected, we performed the secondary hormonal stimulation in permeabilized cells. GrH2 cells were treated with hormone for 1 h, withdrawn from hormone for 20 min, and then permeabilized and washed extensively. Hormone was then added along with cytosol from receptor-negative HeLa cells, ATP, and an energyregenerating system, and the permeabilized cells were incubated for 20 min at 30°C. We found that cytosol and ATP were required to prevent the quantitative association of GRs with the nuclear matrix that occurred upon in vitro 30°C incubation of permeabilized cells in buffer alone (not shown). If hormone was not included upon the incubation of permeabilized cells with cytosol and ATP, GRs remained nuclear yet were extractable by Hypo buffer (Fig. 5, A and B). However, under an identical incubation conducted in the presence of hormone, nuclear GRs regained their resistance to Hypo buffer extraction (Fig. 5, C and D), indicating that the receptors were now tightly bound to chromatin. Therefore, nucleocytoplasmic shuttling of GR includes not only a nuclear to cytoplasmic cycle (Orti et al., 1989; DeFranco et al., 1995; Sackey et al., 1996), but also a nuclear mini-cycle that permits receptors that release from chromatin to reassociate with chromatin upon hormone binding without an obligatory passage through the cytoplasm.


Subnuclear trafficking of glucocorticoid receptors in vitro: chromatin recycling and nuclear export.

Yang J, Liu J, DeFranco DB - J. Cell Biol. (1997)

In vitro recycling of nuclear GRs. GrH2 cells grown on  coverslips were treated with corticosterone (Cort) for 1 h, and  then withdrawn from hormone for 30 min. Cells were then permeabilized and intact nuclei were incubated with 50 μl transport  mixture containing HeLa cytosol, ATP, and an ATP-regenerating system for 20 min at 30°C, with (C and D) or without (A and  B) 1 μM corticosterone present. Nuclei were then either directly  fixed without extraction (A and C) or extracted with Hypo buffer  before fixation (B and D). GRs were detected by IIF.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139874&req=5

Figure 5: In vitro recycling of nuclear GRs. GrH2 cells grown on coverslips were treated with corticosterone (Cort) for 1 h, and then withdrawn from hormone for 30 min. Cells were then permeabilized and intact nuclei were incubated with 50 μl transport mixture containing HeLa cytosol, ATP, and an ATP-regenerating system for 20 min at 30°C, with (C and D) or without (A and B) 1 μM corticosterone present. Nuclei were then either directly fixed without extraction (A and C) or extracted with Hypo buffer before fixation (B and D). GRs were detected by IIF.
Mentions: Since GR nuclear export is extremely slow, we did not expect considerable redistribution of receptors from the nucleus to the cytoplasm during the 20-min hormone withdrawal and 10-min secondary hormone treatment. However, to exclude the possibility that the redistribution of nuclear GRs under our limited hormone withdrawal (i.e., 20 min) was proceeding undetected, we performed the secondary hormonal stimulation in permeabilized cells. GrH2 cells were treated with hormone for 1 h, withdrawn from hormone for 20 min, and then permeabilized and washed extensively. Hormone was then added along with cytosol from receptor-negative HeLa cells, ATP, and an energyregenerating system, and the permeabilized cells were incubated for 20 min at 30°C. We found that cytosol and ATP were required to prevent the quantitative association of GRs with the nuclear matrix that occurred upon in vitro 30°C incubation of permeabilized cells in buffer alone (not shown). If hormone was not included upon the incubation of permeabilized cells with cytosol and ATP, GRs remained nuclear yet were extractable by Hypo buffer (Fig. 5, A and B). However, under an identical incubation conducted in the presence of hormone, nuclear GRs regained their resistance to Hypo buffer extraction (Fig. 5, C and D), indicating that the receptors were now tightly bound to chromatin. Therefore, nucleocytoplasmic shuttling of GR includes not only a nuclear to cytoplasmic cycle (Orti et al., 1989; DeFranco et al., 1995; Sackey et al., 1996), but also a nuclear mini-cycle that permits receptors that release from chromatin to reassociate with chromatin upon hormone binding without an obligatory passage through the cytoplasm.

Bottom Line: Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality.If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited.Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, University of Pittsburgh, Pennsylvania 15260, USA.

ABSTRACT
We have used digitonin-permeabilized cells to examine in vitro nuclear export of glucocorticoid receptors (GRs). In situ biochemical extractions in this system revealed a distinct subnuclear compartment, which collects GRs that have been released from chromatin and serves as a nuclear export staging area. Unliganded nuclear GRs within this compartment are not restricted in their subnuclear trafficking as they have the capacity to recycle to chromatin upon rebinding hormone. Thus, GRs that release from chromatin do not require transit through the cytoplasm to regain functionality. In addition, chromatin-released receptors export from nuclei of permeabilized cells in an ATP- and cytosol-independent process that is stimulated by sodium molybdate, other group VI-A transition metal oxyanions, and some tyrosine phosphatase inhibitors. The stimulation of in vitro nuclear export by these compounds is not unique to GR, but is restricted to other proteins such as the 70- and 90-kD heat shock proteins, hsp70 and hsp90, respectively, and heterogeneous nuclear RNP (hnRNP) A1. Under analogous conditions, the 56-kD heat shock protein, hsp56, and hnRNP C do not export from nuclei of permeabilized cells. If tyrosine kinase inhibitors genistein and tyrphostin AG126 are included to prevent increased tyrosine phosphorylation, in vitro nuclear export of GR is inhibited. Thus, our results are consistent with the involvement of a phosphotyrosine system in the general regulation of nuclear protein export, even for proteins such as GR and hnRNP A1 that use distinct nuclear export pathways.

Show MeSH
Related in: MedlinePlus